枣疯植原体免疫优势膜蛋白基因imp-DZ的克隆与原核表达  

Cloning and prokaryotic expression of imp-DZ gene encoding immunodominant membrane protein from jujube witches’broom phytoplasma

作　　者：董雅容 杨静[1] 宁钧晖 王进忠[1] 王合 田国忠[3] 任争光[1] DONG Yarong;YANG Jing;NING Junhui;WANG Jinzhong;WANG He;TIAN Guozhong;REN Zhengguang(Key Laboratory of Urban Agriculture(North China),Ministry of Agriculture and Rural Affairs,Beijing University of Agriculture,Beijing 102206,China;Beijing Forestry Protection Station,Beijing 100029,China;Research Institute of Forest Ecology,Environment and Protection,Chinese Academy of Forestry,Beijing 100091,China)

机构地区：[1]北京农学院农业农村部华北都市农业重点实验室,北京102206 [2]北京市林业保护站,北京100029 [3]中国林业科学研究院森林生态环境与保护研究所,北京100091

出　　处：《北京农学院学报》2021年第1期22-26,共5页Journal of Beijing University of Agriculture

基　　金：北京市自然科学基金项目(6182002);北京市科技计划项目(Z191100004019025);北京市农业科技项目(20200131);北京市自然科学基金与北京市教育委员会科技计划重点项目(KZ201810020026)。

摘　　要：【目的】为了解枣疯植原体免疫优势膜蛋白的特征,并进行初步表达。【方法】对北京农学院农业农村部华北都市农业重点实验室测得的枣疯植原体JWB-Dongzao免疫优势膜蛋白基因imp-DZ序列进行一致性、进化树和蛋白信号肽与跨膜区分析,分别设计引物扩增完整imp-DZ序列和去除跨膜区编码序列,克隆到蛋白表达载体pBM30上进行原核表达。【结果】imp-DZ基因序列大小为459 bp,编码152个氨基酸,除与枣疯植原体Jwb-nky中一个编码假定蛋白的基因一致性达到96%之外,与其他植原体imp基因核苷酸和氨基酸序列一致性均低60%。枣疯植原体JWB-Dongzao的imp-DZ与16SrV组其他成员imp基因聚在一个分支。携带Imp-DZ跨膜区的重组菌无法表达该蛋白,去掉跨膜区后蛋白得到大量表达。【结论】该研究成功表达了枣疯植原体的免疫优势膜蛋白Imp-DZ,证实了跨膜区影响其在大肠杆菌中的表达,为下一步制备该蛋白抗血清和探索植原体与寄主植物互作机制提供依据。【Objective】This study aims to explore the characteristics of the immunodominant membrane protein(Imp)from jujube witches’broom(JWB)phytoplasma and to induce expression in Escherichia coli.【Methods】The identity and genetic evolutionary relationship of imp-DZ gene from JWB-Dongzao phytoplasma with other imp genes were analyzed,and the signal peptide,transmembrane region of Imp-DZ were also analyzed.The imp-DZ gene was cloned with or without transmembrane region coding sequence to pBM30 vector by designing different primers and heterologously expressed in E.coli.【Results】The size of imp-DZ gene was 459 bp,coding 152 amino acids.Excepting one hypothetical protein gene from Jwb-nky phytoplasma with sequence identity 96%,the identity of nucleotide and amino acid of imp-DZ with other imp genes were less than 60%.The phylogenetic analysis indicated that imp genes from 16SrV group phytoplasma were assigned to a clade.The prokaryotic expression results showed that the Imp-DZ protein couldn’t be heterologously expressed unless the transmembrane region was removed.【Conclusion】In this study,the immunodominant membrane protein Imp-DZ was successfully expressed,and transmembrane region was proved to influence its expression in E.coli.All the results provide bases for further studies on preparing antiserum and exploring the interaction mechanism of phytoplsama and its host plant.

关 键 词：枣疯病 植原体 免疫优势膜蛋白 异源表达 

分 类 号：S436.6[农业科学—农业昆虫与害虫防治]